Protocol 27022: Standard PCR Assay - Generic mCherry
Version 6.2

Notes

This assay will NOT distinguish hemizygous from homozygous transgenic animals.

The genotyping protocol(s) presented here have been optimized for reagents and conditions used by The Jackson Laboratory (JAX). To genotype animals, JAX recommends researchers validate the assay independently upon receipt of animals into their facility. Reaction cycling temperature and times may require additional optimization based on the specific genotyping reagents used.

Expected Results

Transgene = 288 bp

Internal positive control = 200 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
9703 AGG ACG GCG AGT TCA TCT AC Transgene Forward A mCherry
9704 TGG TGT AGT CCT CGT TGT GG Transgene Reverse A mCherry
oIMR8744 CAA ATG TTG CTT GTC TGG TG Internal Positive Control Forward A
oIMR8745 GTC AGT CGA GTG CAC AGT TT Internal Positive Control Reverse A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
9703 0.50 uM
9704 0.50 uM
oIMR8744 0.50 uM
oIMR8745 0.50 uM
Glycerol 6.50 %
Dye 1.00 X
Kapa 2G HS taq polym 0.03 U/ul
DNA

Cycling

Step Temp °C Time Note
1 94.0 --
2 94.0 --
3 65.0 -- -0.5 C per cycle decrease
4 68.0 --
5 -- repeat steps 2-4 for 10 cycles (Touchdown)
6 94.0 --
7 60.0 --
8 72.0 --
9 -- repeat steps 6-8 for 28 cycles
10 72.0 --
11 10.0 -- hold
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.
JAX uses a 'touchdown' cycling protocol and therefore has not calculated the optimal annealing temperature for each set of primers.

Strains Using This Protocol

Stock Number Strain Name
029540 B6.129P2(Cg)-Cd19tm1.1Oao/J
028624 B6.Cg-S1pr3tm1.1Hrose/J
025525 B6.Cg-Tg(Cx3cl1/mCherry)1Jung/J
030748 B6.Cg-Tg(Krt18-RFP)1Jsaw/J
030745 B6.Cg-Tg(Krt5-ECFP)1Jsaw/J
031693 B6.FVB-Tg(CAG-EGFP,-Sumo1,-Sumo2,-Sumo3,-mCherry)10Weiy/J
029899 B6;129S6-Gt(ROSA)26Sortm2(CAG-NuTRAP)Evdr/J
025406 B6;FVB-Tg(RP23-370F21-GCaMP3*/mCherry)1Mik/J
024046 C57BL/6-Tg(Csf1r-HBEGF/mCherry)1Mnz/J
021423 C57BL/6J-Tg(Dlx2-cre,-mCherry)9Grsr/GrsrJ
021853 FVB-Tg(Myh6-mCherry/Map1lc3a)1Rag/J
018067 FVB-Tg(Prism)1849Htz/J
018071 FVB-Tg(Prism)1861Htz/J
029429 STOCK Olfr151tm26Mom Tg(Olfr151,-MAPT/mCherry)5MGaFeins/J
014177 STOCK Tg(Afp-mCherry)1Hadj/J
025693 STOCK Tg(CAG-GFP,-mCherry/Pxn)#Mgin/KleyJ
029600 STOCK Tg(Olfr151-OR1A1,-MAPT/mCherry)D736Feins/J
017470 STOCK Tg(Tnc-mCherry)2Pmay/J
011108 STOCK Tg(Ttr-RFP)1Hadj/J
19 strains use this protocol